A single errant cell can instigate cancer. To trigger this disease, mutant proteins either singly or in groups disrupt normal cellular function. What does one abnormal protein look like? How do the dynamics of the mutant compare to the kinetics of wild- type? In the studies proposed here, single molecules will be individually examined to characterize the basis for their contributions to molecular disease. The microscope used to examine the proteins one-at-a-time is a new type of nanocircuit reported by the Investigators recently in Science. The project leverages advances in microfabrication and the controlled synthesis of a single carbon nanotube contacting multiple electrodes. In published preliminary results, the Investigators have demonstrated conductance-controlled introduction of a single, carboxylate handle onto the sidewall of a nanotube connected into a nanocircuit. Through bioconjugation to the carboxylate handle, a single protein can be connected into the nanocircuit. Though standard EDC/NHS coupling chemistry provides stochastic conjugation to a random lysine, specific cysteine free thiols can be used to direct connections to particular sites within the protein. Using the electronic signature of the resultant nanocircuit, the single protein will be examined in real-time during protein unfolding, folding, binding, and, where applicable, catalysis. In Specific Aim 1, the current design for carbon nanocircuits will be extended for sensitive measurements with multiple proteins in parallel. Single molecule experiments will benefit from this parallel device architecture in two scenarios explored in the next specific aims. Simultaneous interrogation of different proteins or protein variants can elucidate functional differences under identical conditions, such as the abnormality of a mutant protein versus wild-type. In the next specific aim, the carbon nanocircuits from Specific Aim 1 are first applied to investigate well studied proteins, thus establishing a baseline for the approach. Single molecule enzymology will explore how electron transfer, conformational change, allostery, and other issues affect nanocircuit conductance. Specific Aim 3 extends device architectures from the first and what is learned from the second to investigate the molecular basis for caveolin control over cell signaling, implicated in cancer and other diseases. The proposed studies examine how caveolin inhibits different enzymes under a range of different conditions and mutational variants. In summary, given the importance of single molecule events to disease instigation and propagation, expanded methods for single molecule studies are needed. This application leverages recent advances from the Investigators laboratories to develop a generalizable approach for single molecule enzymology. Then, the mechanistic basis for caveolin mediation of cancer will be explored at the single molecule level. PUBLIC HEALTH RELEVANCE Individual proteins can hijack cells to cause cancer and other human diseases. This project develops new technologies for watching individual proteins. Specifically, how caveolin directs tumor formation will be investigated using a new type of nanometer-scale electronic circuit.